Seven million people suffer bone fractures in the U.S. each year. Musculoskeletal conditions cost the U.S. more than $200 billion annually. These numbers are increasing rapidly as the population ages. Human embryonic stem cells (hESCs) offer unlimited supplies of stem cells with a high potential for bone regeneration. However, there has been no report on the use of hESCs for bone tissue engineering via injectable calcium phosphates. While human bone marrow mesenchymal stem cells (hBMSCs) are useful, their harvest requires an invasive procedure, and their proliferation and differentiation potential is lot due to aging and diseases. Therefore, the objectives of this project are to: (1) investigate hESCs in injectable calcium phosphate cement (CPC) for bone engineering, in comparison with human umbilical cord MSCs (hUCMSCs) and hBMSCs; (2) establish the first knowledge base on hESC interactions with CPC scaffolds to guide hESCs for proliferation and osteodifferentiation; (3) design a RGD-grafted CPC scaffold for hESC encapsulation to enhance bone regeneration. While the Arg-Gly-Asp (RGD) peptide has been used in other scaffolds, there has been no report on its use in CPC. Aim 1 will investigate in vitro the hESC encapsulation and differentiation in injectable and macroporous CPC-RGD constructs, and test these hypotheses: (1) hESC-derived MSCs encapsulated in degradable microbeads and incorporated into CPC will synthesize the most amount of bone matrix, followed by hUCMSCs. The gold-standard hBMSCs will make the least bone matrix; (2) RGD-grafted CPC will greatly enhance cell function and bone matrix synthesis, without compromising the CPC injectability and the mechanical properties, which will match the reported strength of cancellous bone. Aim 2 will investigate the hESC-CPC-RGD constructs for bone regeneration in animal model, and test these hypotheses: (1) New bone volume, mineral density, and blood vessel density generated by CPC-RGD with hESC-derived MSCs will be the highest, followed by hUCMSCs. Both of them will far exceed those generated by hBMSCs; (2) hESC-CPC- RGD will be completely replaced by new bone across the entire critical-sized cranial defect at six months; (3) hESC-CPC-RGD will induce much more new bone than that without RGD. This project will yield ground- breaking knowledge on hESC encapsulation in CPC, hESC interaction with RGD-CPC and the guidance of hESCs for osteodifferentiation, and bone regeneration in animal model comparing hESCs, hUCMSCs, and hBMSCs side-by-side. The new stem cell paste can be used in minimally-invasive surgeries, fill complex- shaped defects, and be easily shaped for esthetics in dental and craniofacial applications. The novel hESC- CPC-RGD construct is expected to have wide orthopedic and craniofacial applications, with greatly enhanced bone regeneration to improve the health and quality of life for millions of people. If indeed, hESCs delivered via CPC are superior in osteogenesis compared to the gold-standard hBMSCs, which is anticipated to be shown by this project for the first time, the results will broadly impact the regenerative medicine field.